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Sample GSM4798286

Query DataSets for GSM4798286

Status

Public on Dec 01, 2022

Title

ahtet_plus4hr_PK_notag_2

Sample type

SRA

Source name

S.pombe cells

Organism

Schizosaccharomyces pombe

Characteristics

genotype: plus_ahtet_4hr
chip antibody: Mouse anti V5-Tag (MCA1360, Biorad)
treatment: treated with anhydrotetracycline (ahTet) for 4hr

Growth protocol

S. pombe: 1) For Hi-C experiments, yeast cells were grown to mid-log phases at 32 degrees, unless indicated otherwise. 2) For ChIP-seq in tetracycline-regulated system, cells were grown to mid-log phase at 30 degress in YES medium supplied with 2.5 ug/ml of anhydrotetracycline (ahTet). Depletion of rrp6+ was achieved by culturing cells in YES medium that lacks ahTet at indicated times (8, 12hr). Recovery of rrp6+ was achieved by culturing cells at fresh YES medium containing 2.5ug/ml of ahTet. 3) for ChIP-seq experiments, yeast cells were grown to mid-log phases at 30 degrees, unless indicated otherwise.

Extracted molecule

genomic DNA

Extraction protocol

S. pombe (ChIP) : Fixed cells were sonicated twelve times (15 sec on, 180 sec off) using a Fisher Scientific sonic Dismembrator Model 500 sonicator at 30% output. The immunoprecipitated DNA was eluted using SigmaPrep™ spin column and then subjected to protease treatment and cross-linking reversal. Subsequently, the DNA was purified using Qiaquick PCR purification kit (Qiagen).
S. pombe (RNA isolation): DNase-treated mRNA was purified using NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB), and subjected to library preparation using NEXTflex™ Rapid Directional mRNA-Seq Kit (BIOO) following the manufacturer’s instructions.
S.pombe (in situ Hi-C): formaldehyde-fixed cells (1.6 x 10^7 cells) were digested with zymolyase. Restriction enzyme digestion (MboI – 4 cutter), filling-in, ligation, crosslink reversal, DNA shearing and library construction were done according to previous publications (Rao, SSP et al 2014) with minor modifications.
ChIP-seq libraries for genome-wide sequencing were prepared using either a NEXTflex™ Illumina ChIP-Seq Library Prep kit (5143-02, BIOO) or Accel-NGS 2S Plus DNA Library Kit (21024, SWIFT). mRNA-seq libraries were made using NEXTflex™ Rapid Directional mRNA-Seq kit (5138-10, BIOO) from purified mRNA.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NovaSeq 6000

Description

ahtet_plus4hr_aPK_notag_rpm.bw

Data processing

ChIP-seq samples were sequenced on either Hiseq2500 with single-end method (50-bp reads) or Novaseq6000 with pair-end method (150-bp reads). S.pombe Hi-C samples were sequenced on Hiseq4000 with pair-end method (100-bp reads).
ChIP-seq reads were aligned to the S.pombe genome (ASM294v2) using NovoAlign (V3.04.04, Novocraft technologies) with default parameters. Aligned reads were converted into BAM and replicates were merged using samtools (v1.10). Significantly enriched peaks were identified using MACS2 (v.2.1.4) and mapped reads were quantified using Deeptools (v3.1.3) and bwtool (v1.0).
For mRNA-seq analysis, raw reads were aligned to S. pombe genome (ASM294v2) using STAR aligner. Strand-specific signal tracks of RNA-seq were generated by using bam2wig.py from RseQC (v3.0.1).
Hi-C data were aligned to the S.pombe genome (ASM294v2) using bowtie2 and normalized using HiC-Pro (v2.11.0-beta). The raw and ICE-normalized matrix was generated at 1-kb and 2.5-kb bin resolution using valid interaction pairs.
Genome_build: S. pombe EF2 genome assembly; ASM294v2
Supplementary_files_format_and_content: ChIP-seq: Yeast ChIP-seq bigwig files were created using deeptools bamCoverage (RPM normalization). In case of calibrated bigWigs for set4 experiments, samtools flagstat was used to count reads that were mapped to s.pombe and s.cerevisiae respectively. The occupancy ratio (OR) was calculated according to the previous publication (Hu, B. et al 2015). Then the calibrated s.pombe ChP (Rad21-5FLAGand notag) bigwigs were created using bamCoverage –scaleFactor option; Hi-C: ICE normalized HiC matrix text files and abs files (in HiC-Pro format); RNA-seq: bigwig files and normalized forward/reverser RPKM test files. The number of mapped reads was further analyzed using HOMER and DESeq.

Submission date

Sep 22, 2020

Last update date

Dec 01, 2022

Contact name

Yoonjung Choi

E-mail(s)

[email protected]

Organization name

KAIST

Street address

291 Daehak-ro, Yuseong-gu